Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine (Ser/Thr) kinase that functions to phosphorylate and activate other Ser/Thr kinases in the AGC kinase family (see, e.g., Vanhaesebroeck, B. and D. R. Alessi, “The PI3K-PDK1 connection: more than just a road to PKB,” Biochem. J. (2000), Vol. 346 (Pt 3), pp. 561–76). The best-characterized substrate of PDK-1 is the intracellular Ser/Thr kinase known as AKT, whose expression and/or activity is elevated in many cancers. Kinase activity of serum and glucocordicoid regulated kinase (SGK), which is structurally related to AKT, can also be phosphorylated and activated by PDK-1. Once activated in tumors, AKT promotes increased tumor cell survival, drug resistance, growth and angiogenesis. Three highly related isoforms of AKT, termed AKT1, AKT2 and AKT3 are known in humans. Activation of AKT is dependent on the activity of phosphatidylinsoitol-3 kinase (PI-3 kinase), whose activity is activated by many signaling molecules elevated in cancer cells, including growth factor receptors (e.g., epidermal growth factor (EGF) receptor, ErbB2 and IGF1-receptor) and oncogenes (e.g, Ras, BCR-abl, and Src). Other potential substrates of PDK-1 include p70 S6 kinase, p90 S6 kinase, protein kinase C, cAMP-dependent protein kinase (PKA), PRK1, Protein kinase G and serum and glucocorticoid regulated kinase (SGK).
PDK-1-mediated phosphorylation of AKT, which is largely present in an inactive form in unstimulated cells, converts the enzyme to a catalytically active form. This occurs through the phosphorylation of the activation loop domain of AKT, e.g., at threonine-309 in AKT2 and theonine-308 in AKT1. Phosphorylation of a homologous domain in many kinases is known to regulate their kinase activity. One stimulus for PDK-1-mediated phosphorylation of AKT is the association PI-3 kinase products, (3,4,5)PIP3 or (3,4)PIP2, with the pleckstrin homology (PH) domain of AKT. Although AKT displays low, basal levels of activation in normal, unstimulated cells, AKT often becomes constitutively activated in tumor cells. This occurs through the up-regulation of a variety of different signaling molecules or the presence of oncogenenic mutations commonly found in cancer cells that can promote the activation of AKT, such as PI-3 kinase, growth factor receptors (e.g., EGFR family members), Ras, Src, and BCR-ABL activation. Loss of the tumor suppressor PTEN is another means of greatly increasing AKT activity in cancer cells (Besson, A. et al., “PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis,” Eur. J. Biochem. (1999), Vol. 263, No. 3, pp. 605–611). PTEN mutation or down regulation of PTEN protein is found in a large number of tumors and cancer cell lines. PTEN is a phosphatase that removes the D-3 phosphate from the products of PI-3 kinase such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (Myers, M. P. et al., “The lipid phosphatase activity of PTEN is critical for its tumor supressor function,” Proc. Natl. Acad. Sci. USA (1998), Vol. 95, No. 23, pp. 13513–13518; Stambolic, V. et al., “Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN,” Cell (1998), Vol. 95, No. 1, pp. 29–39). Loss of PTEN, therefore, has the effect of increasing products of PI-3 kinase and promoting constitutive activation of AKT. Cancers with highly up-regulated levels of AKT may be especially sensitive to the effects of PDK-1/AKT pathway inhibitors.
Downstream substrates of PDK-1 and/or AKT are associated with a number of cell responses including proliferation, metabolism and cell survival (Testa, J. R. and A. Bellacosa, “AKT plays a central role in tumorigenesis,” Proc. Natl. Acad. Sci. USA (2001), Vol. 98, No. 20, pp. 10983–5; Vivanco, I. and C. L. Sawyers, “The phosphatidylinositol 3-Kinase AKT pathway in human cancer,” Nat. Rev. Cancer (2002), Vol. 2, No. 7, pp. 489–501). Examples of signaling molecules downstream from PDK-1 or AKT involved in these pathways include BAD, p70 S6 kinase, p21 (Waf-1/Cip-1), Forkhead transcription factors, p27 (kip-1), GSK-3-alpha/beta, TSC2 (tuberin), and ecNOS. The survival function of AKT is particularly well-characterized cellular activity of AKT (Datta, S. R. et al., “Cellular survival: a play in three Akts,” Genes Dev. (1999), Vol. 13, No. 22, pp. 2905–27). AKT functions to suppress cell death, i.e., apoptosis, induced by a variety of agents, including UV radiation, chemotherateutic drugs, TFG-beta, withdrawal of survival factors, overexpression of oncogenes such as c-myc and detachment of cells from the extracellular matrix.
The ability to escape apoptosis is a critical characteristic of tumor cells allowing their uncontrolled growth and invasive behavior. One trigger for apoptosis is the perturbation of the normal growth regulation resulting from oncogenic mutations or inappropriate expression signaling molecules coupled to cell proliferation. Apoptotic pathways, therefore, provide a key means of protection from the development and progression of cancer. Cancer cells, however, can escape apoptotic death by selecting for activation of signaling molecules such as AKT that turn off apoptotic signals. Some oncogenes, such as Ras, which is activated in as many as 60% of human tumors, simultaneously promote uncontrolled growth and the activation of AKT. Inhibition of AKT in NIH 3T3 cells prevents transformation of these cells through transfection with activated Ras. Furthermore, a number of studies have shown that combining expression of an oncogene with an activated form of AKT greatly facilitates formation of tumors in vivo (e.g., Holland, E. C. et al., “Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice,” Nat. Genet. (2000), Vol. 25, No. 1, pp. 55–7). Inhibitors of PDK-1, by blocking activation of AKT, are therefore a means of promoting apoptosis in tumors cells, especially, but not necessarily limited to those over-expressing AKT activity.
Inhibitors of the PDK-1/AKT pathway are also expected to block cancer progression through inhibition of tumor-stimulated angiogenesis (Dimmeler, S. and A. M. Zeiher, “Akt takes center stage in angiogenesis signaling,” Circ. Res. (2000), Vol. 86, No. 1, pp. 4–5; and Shiojima, I. and K. Walsh, “Role of Akt signaling in vascular homeostasis and angiogenesis,” Circ. Res. (2002), Vol. 90, No. 12, pp. 1243–50). AKT has been shown to regulate a number of responses critical for the process of angiogeneisis, including endothelial cell migration, proliferation and survival during new vessel formation, ecNOS regulation, response of endothelial cells to growth factors (including IGF-1, agniopoetin-1 and VEGF) and the regulation of hypoxia-inducible factor-1 (HIF-1)-alpha levels.
Inhibition of the cell cycle and growth of tumor cells is yet another expected effect of compounds that block PDK-1 and/or AKT. Inhibition of PDK-1 and/or AKT activity has been shown to regulate growth of cancer cells in a number of studies. These effects may occur through PDK-1 or AKT-mediated regulation of a number of different signaling pathways important in growth regulation. For example, AKT has been shown to block nuclear localization and/or expression of the cyclin-dependent kinase inhibitors, p21 (Waf-1/Cip-1) and p27 (kip-1). Inhibitors blocking these effects would be expected to reduce the activity of cyclin-dependent kinases, blocking progression through the cell cycle and reducing tumor cell growth. AKT was found to inhibit Myt1, thereby acting as an initiator of mitosis in oocytes from the starfish Asterina pectinfera. Furthermore, PDK-1 and/or AKT regulate the expression of proteins important for cell growth through its regulation of mTOR, p70 S6 kinase and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). While the mechanism of this regulation is not firmly established, it has been shown that AKT phosphorylations reduces expression of TSC2, thereby relieving TSC-2 mediated suppression of mTOR activity. This, in turn, promotes the activation p70 S6 kinase activity and the phosphorylation and inhibition of 4E-BP1 (Inoki, K. et al., “TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling,” Nat. Cell. Biol. (2002), Vol. 12, p. 12; and Potter, C. J. et al., “Akt regulates growth by directly phosphorylating Tsc2,” Nat. Cell. Biol. (2002), Vol. 12, p. 12). Both these effects result in increased synthesis of mRNAs encoding proteins important for cell growth. Loss of TSC2 function is associated with the disease tuberous sclerosis, which results in differentiated benign growths (harmatomas) in a wide variety of organs. PDK-1 also has been shown to have a direct role in the phosphorylation and activation p70 S6 kinase (Alessi, D. R. et al., “3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro,” Curr. Biol. (1998), Vol. 8, No. 2, pp. 69–81).
Compounds which block PDK-1 mediated activation of AKT or PDK-1 directly may therefore be useful therapeutic agents in treating a variety of disease-states, such as cancer.